The present invention relates generally to microscopy and more specifically to stereoscopic confocal microscopy.
The art of microscopy deals with the development of methods and instruments for magnifying. A significant portion of microscopy deals with using light from the visible portion of the spectrum to illuminate the sample to be magnified. This aspect of the art of microscopy is known as light microscopy.
It is well understood that many factors influence the maximum attainable axial and lateral resolution using light microscopy techniques. One of these limiting factors is the field size. Specifically, when the field of view of a lens is extremely small, the axial and lateral resolution of the image being magnified can in fact be greater than when the field of view of the lens is broad (or not limited). These theoretical considerations set the stage for the development of an apparatus known as the confocal microscope.
In U.S. Pat. No. 3,013,467 (hereinafter '467) issued on Dec. 19, 1961 to M. Minsky (see FIG. 1), a confocal microscope is disclosed. In this patent, Minsky discloses specimen 22 mounted upon the reflective surface of mirror 15. A beam splitting plate 17 is interposed between the collimating plate or wall 14 and lens 11. The reflective surface of beam splitting plate 17 faces lens 11, while the transparent surface of plate 17 faces pinhole aperture 16.
The light reflected from bulb 10 by reflector 12 is collimated by pinhole aperture 16 of plate 14 to provide a point source of light A. Divergent beam B,B passes through beam splitting plate 17 and then through lens 11 becoming convergent beam C,C. Focal point D of beam C,C is located on specimen 22 and becomes divergent beam E,E which is reflected from mirror 15 back through lens 11. Lens 11 forms convergent beam F,F which is reflected perpendicularly from beam splitting plate 17 as indicated by beams F',F' which converge to their focal point G at pinhole aperture 26 of plate 24. Photoelectric cell 28 is located in alignment with aperture 26 to measure the intensity of the light passing therethrough. Because pinhole apertures 16 and 26 lie upon the optical axis OA of the confocal microscope of FIG. 1, the point source of light A and the specimen point of illumination D both originate on optical axis OA, while the point image G terminates on the optical axis OA. Therefore, all of the light rays accepted by photoelectric cell 28 must pass through specimen 22 at point D on optical axis OA and pass again through optical axis OA at point G. Light scattered from points other than point D of specimen 22 is, for the most part, rejected from the optical system. Such scattered rays may pass through and be refracted by lens 11 but will not be directed to pinhole aperture 26. Rather, this scattered light will strike the body of plate 24 and be rejected from the optical system. Such rays can re-enter the optical system only by again being scattered, and the possibility of this scattering taking place along a line through point D on optical axis OA is remote. Pinhole aperture 26 increases the optical resolution of the system by its action of squaring the intensity pattern distribution of the image defraction.
Because the confocal microscope of FIG. 1 provides a high degree of selectivity, the following advantages are gained:
(1) minimum image blurring, PA1 (2) increase in signal-to-noise ratio, PA1 (3) increase in effective resolution, PA1 (4) high resolution light microscopy through unusually thick and highly scattering specimens, and PA1 (5) very narrow depth of focus.
As was discussed above in conjunction with the prior art confocal microscope of FIG. 1, the optical system disclosed therein brings into focus the light originating at a single point on the optical axis. Thus, if one desires to, look at several portions of sample 22, some means must be provided to move the point of specimen illumination D. Minsky discloses one such means in his '467 patent which involves continually moving (or scanning) sample 22 relative to the optical system. Although this technique works satisfactorily for small samples, larger samples cannot be easily adapted to this type of scanning and alternative methods are used.
One alternative to the above-mentioned method of moving the sample relative to the optical system, involves the technique of moving (or scanning) specimen illumination point D relative to sample 22. Although several different techniques may be used to accomplish the scanning of illmunination point D, a popular technique involves using a spinning opaque wheel 90 (also known as a Nipkow disk) that is perforated by a series of apertures 92. Apertures 92 can be in the form of pinholes (as shown in FIG. 2A) but other aperture geometries are also useable (such as slits). The successive holes are placed at a constant angle apart 94 around the center of disk 90 but on a constantly decreasing radius 96 (i.e. arranged as an Archimedes spiral). The basic idea behind the Nipkow disk is instead of using a single pinhole 16 in plate 14 and moving sample 22, a large number of pinholes are placed in plate 14 thereby providing a means of scanning specimen illumination point D while maintaining specimen 22 stationary. Pinholes 92 are sufficiently separated so that there is no interaction between the images formed by the individual pinholes. The complete image is formed by moving the pinholes so as to fill the space in between them. Typically, moving the pinholes involves rotating disk 90 about its center. The pinhole arrangement seen in the prior art Nipkow disk of FIG. 2A would produce a raster scan pattern across sample 22 as depicted in FIG. 2B.
Although the basic concepts of confocal microscopy are understood and documented (for example see generally Handbook of Biological Confocal Microscopy, edited by James B. Pawley, Integrated Microscopy Resource for Biomedical Research University of Wisconsin-Madison, Madison, Wis., revised edition, Plenum Press, New York and London, specifically see Chapter 1, Foundations of Confocal Scanned Imaging in Light Microscopy; Chapter 10, Intermediate Optics in Nipkow Disk Microscopes; and Chapter 11, The Role of the Pinhole in Confocal Imaging Systems, also see Physics Today, Sep. 1989, by Gordon S. Kino of Stanford University), current designs continue to be improved upon. For example, notwithstanding the above-mentioned advantages associated with using a Nipkow disk to scan the light source across the sample, a major disadvantage is that the Nipkow disk blocks typically 99% of the illuminating light emunating from bulb 10. This in turn requires the use of a very intense light source (such as an arc lamp or laser). In addition to the above drawback, the use of Nipkow disks also produces a high percentage of reflected light (light which does not pass through the pinhole opening of the disk) which, in turn, causes artifacts in the final image.
A co-pending application Ser. No. 07,734,398 filed Jul. 23, 1991 (hereinafter the '398 application) which is hereby incorporated by reference, sets forth an improved confocal microscope which addresses some of these disadvantages.
One disadvantage which is not addressed is the '398 application is the loss of stereoscopic vision when employing the typical confocal optical microscope.
Stereoscopic viewing is an extremely desirable feature of an optical microscope because it enables the viewer to see the shapes and depth relationships of the various objects in the field of view of the microscope. For low-power microscopes, stereoscopic capability has been implemented (with some degree of success) through the use of conventional optics. However, for high-power (i.e. high magnification microscopes), conventional techniques have been less successful (with the degree of success decreasing proportionate with increasing optical power). The root of this difficulty lies in a fundamental law governing the magnification of any lens, namely that the axial magnification (magnification along the line-of-sight) is always greater than the lateral magnification (magnification perpendicular to the line-of-sight). Specifically, the axial magnification is equal to the square of the lateral magnification. This relationship flows directly from the well-known lens equation. This difference in axial and lateral magnification results in an elongation of the three-dimensional image of the object. This elongation takes place along the axial direction, and (because of the square-law magnification relationship) the degree of elongation increases dramatically with increasing magnifying power of the objective lens. When this elongated image is viewed through the microscopes eyepiece (which necessarily has a finite depth of focus) only a very narrow slice of the image can be in focus (at any given time) for the observer. Consequently, the depth of field (i.e. the in-focus field of view in the axial direction of a conventional high-power microscope) is necessarily restricted to a narrow slice of the three-dimensional object and the sensation of three-dimensionality is largely sacrificed. As a result, the viewer is reduced to the tedious task of observing the object one layer at a time and trying to reconstruct the three-dimensional object from memory after all layers have been observed. Techniques are known for acquiring and storing images at different depths of the object. After the images are acquired and stored, they are reconstructed by projecting them sequentially on a screen which oscillates along the line-of-sight of the viewer. See R. L. Gregory, The Solid Image Microscope, Research 13, Pages 422-427 (1960) and Alan Boyde, Direct Recording Of Steroscopic Pairs Obtained From Disk-Scanning Confocal Light Microscopes, Chapter 13 of the Handbook of Biological Confocal Microscopy, Pages 163-168. Prior art also exists in which the object distance is varied rapidly as a function of time, the result of which is successively bringing different layers of the object into focus (in effect rapidly scanning through the depth of the object). See R. L. Gregory, Procedures of the Second International Conference on Medical Electronics, Paris, France, 1959, edited by C. N. Smyth: London, Iliffe (1960), Page 591. The same effect can be obtained by rapidly oscillating the focusing knob of a conventional microscope.
Although the approach of rapidly oscillating the focusing knob of a conventional microscope does give some sense of three-dimensionality, it is hampered by two problems: (1) the images of the different layers are formed at the same depth of view in the eyepiece so, even though they might be viewed through binocular eyepieces, they are not seen as a three-dimensional image but merely as a succession of two-dimensional images, and (2) the light reflected or scattered (depending on the lighting arrangement) from layers other than the one which is instantaneously ill focus reaches the eyes and presents an annoying out-of-focus background. Because the confocal microscope (by virtue of the attributes which were earlier discussed) has the ability to reject light from layers of the object other than that layer which is currently in focus, its use eliminates the second of the two problems (i.e. the out-of-focus background problem). However, the first enumerated problem (that of achieving the sensation of three-dimensional vision) is not eliminated by virtue of using a conventional confocal microscope.
In low-power stereoscopic microscopes, this first problem is solved by using long focal length objective lenses with small apertures to increase the depth of field for the intermediate image within the microscope. Two objective barrels are used to obtain two views of the intermediate image at slightly different angles for each eye, thus creating the sense of three-dimensionality. This procedure, however, fails for high-power microscopes because their greater magnification cannot be achieved with long focal length objective lenses.
Therefore, there is a need for a high-quality, direct-viewing, high-power, stereoscopic microscope which possesses tile ability of the confocal microscope to reject out-of-focus light and which also possesses the ability to present to the viewer two different angular views of the object.
The present invention achieves these objectives by modifying a conventional confocal microscope. The modified confocal microscope of the present invention operates by sequentially bringing into focus various depths of the object (or specimen) to be viewed. One way of accomplishing this is by varying the specimen distance as in the prior-art microscope described above. This is preferably accomplished by vibrating either the specimen or the objective lens (or some other lens capable of altering the focal plane of the objective lens). Because we are employing a confocal microscope, the out-of-focus background problem is automatically solved. The remaining problem (that of providing a different angular view for each eye) is solved through the introduction of a set of movable prisms (or, alternatively, movable mirrors). These movable prisms (or mirrors) are vibrated in synchronism firstly with one another and secondly with the specimen in such a way as to present images which are at different depths within the specimen and which provide different parallax at the eyes. It is this different parallax which is responsible for the visual sensation of three-dimensionality (both in the real world and in any common stereoscopic viewing system). Devices which have traditionally taken advantage of creating a visual sensation of three-dimensionality by providing different parallax at each eye include the old-fashioned stereoscope and the modern 3-D movies.
One additional problem that is introduced through the use of the confocal principle (which is a problem which that is common to all direct-viewing confocal microscopes) is that of low image brightness. This problem is solved in an alternative embodiment of the present invention through the use of a focusing lens which is placed between the illuminating light source and the pinhole aperture.